Mycobacterium tuberculosis (Mtb) is a major opportunistic pathogen and the leading infectious disease- related cause of death among people living with HIV worldwide. Shorter TB treatment regimens are urgently needed to minimize drug interactions with antiretroviral drugs, improve medical adherence, and curb the emergence of TB drug resistance. The prolonged duration of TB treatment is believed to reflect the unique ability of a subpopulation of bacteria to remain in a non-replicating, persistent state in the infected host. These ?persister? bacteria evade immune-based clearance mechanisms and become tolerant to first-line anti-TB drugs, which more effectively target actively dividing bacteria. One of the important mechanisms by which persisters acquire this phenotype is through induction of the stringent response. The stringent response enzyme RelMtb is required for bacterial growth restriction and antibiotic tolerance, and is essential for long-term Mtb survival during various in vitro and in vivo stress conditions. Deficiency of relMtb renders Mtb more sensitive to isoniazid in various models. We have generated a therapeutic DNA vaccine targeting Mtb stringent response genes, which induces antigen-specific cellular immunity, and, when combined with the first-line drug isoniazid, exhibits significantly greater activity against Mtb in the lungs of chronically infected mice relative to isoniazid alone. We also have developed a novel vaccination strategy involving fusion of the antigen of interest with the dendritic cell-targeting chemokine MIP3?, which significantly enhances immunogenicity. This vaccine platform is designed to address the fact that in addition to the T-cell deficiencies of HIV infection, there is clear documentation of deficiency of DC function in both the TB and HIV settings. However, it remains to be determined if enhanced immunity to Mtb persistence factors can shorten the duration of curative antibiotic treatment in the immune-competent and T-cell-deficient host. In the current proposal, we will directly address these knowledge gaps by testing the novel hypothesis that enhanced cellular immunity against Mtb stringent response factors potentiates the activity of the first-line anti-TB regimen and accelerates cure in the standard murine model of TB. Since HIV infection is associated with disturbed T-cell homeostasis, including depletion of CD4+ T cells and persistent expansion of CD8+ T cells, we will characterize the contribution of each of these cell types to the therapeutic efficacy of the stringent response vaccine in mice. This proposal represents a unique collaboration between Investigators with significant expertise in microbiology, molecular biology, immunology, DNA vaccines, and animal models. Our findings are expected to inform future studies investigating therapeutic TB vaccines, and lay the groundwork for the development of novel strategies to therapeutically target Mtb persisters, with the goal of shortening treatment for drug-susceptible and drug-resistant TB in HIV-infected and uninfected individuals.